Sheep Blow Fly Lucilia Cuprina Research Articles

Building a transgenic sexing strain for genetic control of the Australian sheep blow fly Lucilia cuprina using two lethal effectors

BackgroundThe sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to “transcriptional squelching”, while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain.ResultsHere a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain.ConclusionsCompared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.

Using Moderate Transgene Expression to Improve the Genetic Sexing System of the Australian Sheep Blow Fly Lucilia cuprina.

Simple SummaryPopulations of pest insects can be suppressed through repeated mass releases of sterilized insects. This is particularly effective if only sterile males are released. We previously developed several genetically modified strains of the Australian sheep blowfly that produce only males when raised on diet that lacked tetracycline. A disadvantage of the some of the engineered strains was that females would lay few eggs unless fed a diet with a low dose of tetracycline. In this study we show that effective male-only strains can be made by combining driver/effector lines that have moderate transgene expression/activity. Furthermore, the strain does not require tetracycline in the adult diet for female fertility. This “moderate expression/activity” strategy could be more generally applied to other pests that can be genetically modified.The sterile insect technique (SIT) is a promising strategy to control the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. We have previously developed a transgenic embryonic sexing system (TESS) for this pest to facilitate the potential SIT application. TESS carry two transgenes, a tetracycline transactivator (tTA) driver and a tTA-activated pro-apoptotic effector. TESS females die at the embryonic stage unless tetracycline is supplied in the diet. However, undesired female sterility was observed in some TESS strains without tetracycline due to expression of tTA in ovaries. Here we investigate if TESS that combine transgenes with relatively low/moderate expression/activity improves the fertility of TESS females. tTA driver lines were evaluated for tTA expression by quantitative real time PCR and/or by crossing with a tTA-activated RFPex effector line. Fertility and lethality tests showed that a TESS strain containing a driver line with moderate tTA expression and an effector line showing moderate pro-apoptotic activity could recover the fertility of parental females and eliminated all female offspring at the embryonic stage. Consequently, such a strain could be further evaluated for an SIT program for L. cuprina, and such a “moderate strategy” could be considered for the TESS development in other pest species.

Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters.

For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.

Building early-larval sexing systems for genetic control of the Australian sheep blow fly Lucilia cuprina using two constitutive promoters

Transgenic sexing strains (TSS) that carry conditional female lethal genes are advantageous for genetic control programs based on the sterile insect technique (SIT). It is desirable if females die early in development as larval diet is a major cost for mass production facilities. This can be achieved by using a gene promoter that is only active in embryos to drive expression of the tetracycline transactivator (tTA), the transcription factor commonly used in two-component TSS. While an embryo-specific promoter is ideal it may not be essential for assembling an effective TSS as tTA can be repressed by addition of tetracycline to the diet at larval and/or adult stages. Here we have investigated this idea by isolating and employing the promoters from the Lucilia spitting image and actin 5C genes to drive tTA expression in embryos and later stages. L. cuprina TSS with the tTA drivers and tTA-regulated tetO-Lshid effectors produced only females when raised on a limited tetracycline diet. The Lshid transgene contains a sex-specific intron and as a consequence only females produce LsHID protein. TSS females died at early larval stages, which makes the lines advantageous for an SIT program.

A transgenic embryonic sexing system for the Australian sheep blow fly Lucilia cuprina.

Genetic approaches, including the sterile insect technique (SIT), have previously been considered for control of the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. In an SIT program, females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males, thereby decreasing the efficiency of the program. Consequently, transgenic sexing strains of L. cuprina were developed that produce 100% males when raised on diet that lacks tetracycline. However, as females die mostly at the pupal stage, rearing costs would not be significantly reduced. Here we report the development of transgenic embryonic sexing strains of L. cuprina. In these strains, the Lsbnk cellularization gene promoter drives high levels of expression of the tetracycline transactivator (tTA) in the early embryo. In the absence of tetracycline, tTA activates expression of the Lshid proapoptotic gene, leading to death of the embryo. Sex-specific RNA splicing of Lshid transcripts ensures that only female embryos die. Embryonic sexing strains were also made by combining the Lsbnk-tTA and tetO-Lshid components into a single gene construct, which will facilitate transfer of the technology to other major calliphorid livestock pests.

Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina

The New World screwworm and the Australian sheep blowfly Lucilia cuprina are devastating pests of livestock. The larvae of these species feed on the tissue of the living animal and can cause death if untreated. The sterile insect technique or SIT was used to eradicate screwworm from North and Central America. This inspired efforts to develop strains containing complex chromosomal rearrangements for genetic control of L. cuprina in Australia. Although one field trial was promising, the approach was abandoned due to costs and difficulties in mass rearing the strain. As the efficiency of SIT can be significantly increased if only sterile males are released, we have developed transgenic strains of L. cuprina that carry a dominant tetracycline repressible female lethal genetic system. Lethality is due to overexpression of an auto-regulated tetracycline repressible transactivator (tTA) gene and occurs mostly at the pupal stage. Dominant female lethality was achieved by replacing the Drosophila hsp70 core promoter with a Lucilia hsp70 core promoter-5′UTR for tTA overexpression. The strains carry a dominant strongly expressed marker that will facilitate identification in the field. Interestingly, the sexes could be reliably sorted by fluorescence or color from the early first instar larval stage as females that overexpress tTA also overexpress the linked marker gene. Male-only strains of L. cuprina developed in this study could form the basis for a future genetic control program. Moreover, the system developed for L. cuprina should be readily transferrable to other major calliphorid livestock pests including the New and Old World screwworm.

In Vitro Induction of 60-kDa and 70-kDa Heat Shock Proteins by Endosulphan and Monocrotophos in Sheep Blowfly Lucilia cuprina

The effect of two most commonly used and highly toxic organic pesticides, endosulphan (organochlorine) and monocrotophos (organophosphate), was studied in a blowfly, Lucilia cuprina, to test whether these pesticides induce the stress response and, if so, whether the intensity of the response, in terms of induction of heat shock proteins (HSPs), HSP60 and HSP70 in particular, is pesticide concentration dependent. The in vitro exposure of larval and adult tissues to varying concentrations of these pesticides (endosulphan: 1.0-4.0 ppm for larva and 0.05-0.50 ppm for adult; monocrotophos: 0.0005-0.0050 ppm for larva and 0.0001-0.0010 ppm for adult) revealed that both compounds were able to induce the expression of HSP60 and HSP70 proteins. Western blot analysis of these HSPs indicated that the induction of expression was tissue-specific. The trypan blue staining of pesticide-exposed tissues demonstrated monocrotophos to exert more severe effect than endosulphan, as the former compound induced both HSP60 and HSP70 significantly at a much lower concentration than that of the later. The pattern of expression of these HSPs, in general, appeared in direct correlation with the pesticide concentration. Gut tissues were found relatively more sensitive to pesticide toxicity than other tissues, as revealed by trypan blue staining, and hence, they might serve as primary targets for early detection of pesticide toxicity. The results indicated that either of these HSPs or both could serve as a potential biomarker toward assessment and monitoring of toxicity induced by these pesticides.

Nicholson's blowflies revisited: A fuzzy modeling approach

We apply fuzzy modeling to derive a mathematical model for a biological phenomenon: the regulation of population size in the Australian sheep-blowfly Lucilia cuprina. This behavior was described by several ethologists and fuzzy modeling allows us to transform their verbal descriptions into a well-defined mathematical model. The behavior of the resulting mathematical model, as studied using both simulations and rigorous analysis, is congruent with the behavior actually observed in nature. We believe that the fuzzy modeling approach demonstrated here may supply a suitable framework for biomimicry, that is, the design of artificial systems based on mimicking natural behavior.

Analysis of the circadian clock gene period in the sheep blow fly Lucilia cuprina.

We have isolated a homologue of the period (per) gene from the Australian sheep blow fly, Lucilia cuprina, as part of a comparative approach to the analysis of dipteran circadian systems. Sequence analysis of the 4 kb per cDNA revealed the conservation of three functional domains, namely the PAS dimerization motif, and the nuclear and cytoplasmic localization domains. A fourth domain, the threonine-glycine (TG) repeat region, is also conserved in L. cuprina per but has been severely truncated. No length variation was found in the TG repeat of L. cuprina or L. sericata collected from several different latitudinal zones. Expression analysis indicated a diel oscillation in per mRNA in LD 12:12 with a period of 24 h and a peak at Zt 12. PER-immunoreactive protein oscillations were also demonstrated, with peak immunoreactivity lagging approximately 3 h behind peak mRNA levels. These results show the existence of a Drosophila-like circadian system in a calliphorid fly. They also provide evidence for the conservation of per function across the Diptera, and confirm the relevance of the Drosophila system as a model for fly circadian rhythms.

Characterization of Malathion Carboxylesterase in the Sheep Blowfly Lucilia cuprina

Resistance to malathion in a strain of the Australian sheep blowfly is due to a 10-fold increase in malathion carboxylesterase (MCE) activity relative to a more susceptible strain. MCE was purified to apparent homogeneity from these two strains and was shown to be a monomer of 60,500, with a pI of 5.5 in both strains. Purified MCE from both populations had identical Km and Vmax values for the hydrolysis of malathion as well as for three other esterase substrates. Similarly, the kinetics of inhibition by several inhibitors were the same for the MCE from each strain. These data therefore suggest that resistance to malathion is due to a quantitative rather than a qualitative change in the MCE of the two strains. Estimation of the total MCE content in each strain showed that the resistant blowflies had nine times more MCE than the more susceptible insects. Although blowfly MCE showed greater specificity for naphthyl esters over malathion, it nevertheless hydrolyzes malathion faster than any other esterase yet isolated from an insect. This is in sharp contrast to previously studied insect strains in which organophosphate resistance has been attributed to large increases in nonspecific esterases that show very slow or no hydrolysis of the insecticides.

Physiological Basis of Ratio‐Dependent Predator‐Prey Theory: The Metabolic Pool Model as a Paradigm

Ratio—dependent models of predators and prey are approximations of the biology of resource acquisition and allocation and their consequences for population birth and death rates. A demand—driven functional response model that has a physiological basis in mass (energy) dynamics is reviewed (i.e., the metabolic pool model), and its obvious links to the logistic model are outlined. To demonstrate the utility of this approach, a distributed maturation time age—structure model of the dynamics of A. J. Nicholson's classic laboratory population data on the sheep blow fly (Lucilia cuprina Weidman) is developed. The model provides sufficient information on the dynamics of the intermediate life stages to show that the blow fly oscillations were due to the effects of larval competition for food on size, fecundity, and pupation success. These results agree with Nicholson's conclusions. The advantage of this model, in contrast to prior models, is that the dynamics emerge by considering the processes of resource acquisition and allocation as they affect growth, reproduction, and survival. No explicit time delays, which automatically lead to oscillations, were included. Lastly, the notions of the metabolic pool model are found in Nicholson's original model for equilibrium population density. The metabolic pool paradigm in an age—structure setting is used to model the tri—trophic dynamics of Acyrthosiphon aphids in an alfalfa ecosystem. The model explains the role of the various natural enemies in the regulation of the aphids.

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Clones carrying the white and topaz eye color genes have been isolated from genomic DNA libraries of the blowfly Lucilia cuprina using cloned DNA from the homologous white and scarlet genes, respectively, of Drosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of the white gene of L. cuprina has been determined, and the homology to the corresponding region of D. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing the D. melanogaster genes is free of repeated sequences, that encompassing their L. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome of L. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10(4) copies to less than 10(2) copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both the white and topaz gene regions are found within and between populations of L. cuprina.

Spectral properties of movement perception in the dronefly Eristalis

1. Spectral-sensitivity functions of large-field movement-detecting units in the lobula plate of the dronefly Eristalis tenax L., which is a Batesian mimic of the honeybee, were measured using visual stimuli consisting of light flashes, or moving gratings. Two classes of units were studied, one class responding to inward horizontal motion in the contralateral eye (presumably the homologue of the well-known ‘HI’ in other fly species), and the other class responding to vertically-down-ward motion in the contralateral eye. 2. In both classes of units, the spectral-sensitivity function of the response to ‘flashes’ is characterized by two peaks, one in the UV at ca. 350 nm and the other in the blue at ca. 475 nm (Figs. 3, 8). It resembles the spectral-sensitivity function of the R1-R6 class of receptors in other flies. 3. In both classes of units, the spectral-sensitivity function of the response to ‘movement’ is characterized by a single peak, occurring in the blue at ca. 450 nm (Figs. 7, 9). 4. Control experiments on homologous units in the Australian Sheep Blowfly Lucilia cuprina, using identical stimulating conditions reveal that the response to flashes as well as movement possesses a dual-peaked spectral sensitivity, with one peak in the UV and the other in the blue-green region of the spectrum (Figs. 10–12). 5. The results indicate that the pathways subserving the inputs to movement-detecting neurons in Eristalis are driven by more than one spectral class of photoreceptors. They also reveal that the spectral sensitivity of movement detection in Eristalis bears a closer resemblance to that of the honeybee, than to that of other flies. This similarity to the honeybee may arise from the fact that the dronefly and the honeybee occupy similar ecological niches, both foraging for nectar in flowers.

Genetic analysis of field trials of sex-linked translocation strains for genetic control of the Australian sheep blowfly Lucilia cuprina (Wiedemann).

The results of progeny tests of males and females captured during two field trials of sex-linked translocation strains for genetic control of L. cuprina are presented. Males released as mature larvae survived to adulthood and mated with field females. However, the levels of genetic death introduced into the population were insufficient to suppress the native population. This was due partly to seasonal ineffectiveness of the release method, and partly to poor performance of the released males. On average, the mating competitiveness of the released males was only one-third that of field males, whereas their field-reared, translocation-bearing sons were fully competitive with native males.

Estimation of the Relative Viabilities of Insecticide Resistance Genotypes of the Australian Sheep Blowfly Lucilia cuprina

Artificial strikes were initiated on sheep at different times after treatment with either diazinon (an organophosphorus compound) or lindane (an organochlorine compound).

Effects of Several Larvicidal Compounds on Chitin Biosynthesis by Isolated Larval Integuments of the Sheep Blowfly Lucilia cuprina

Isolated whole integuments from L. cuprina larvae rapidly incorporate radioactivity from both N-acetyl[I-'4C]glucosamine and [1-'4C]glucosamine into alkali-insoluble material, a reaction which does not require preincubation of the tissue with /i-ecdysone. The labelled product was degraded to N-acetylglucosamine during digestion with chitinase, establishing that it consists mainly of chitin. Incorporation was inhibited by polyoxin-D (Iso, 6 x 10- 7 M) and diflubenzuron (Iso, 7 x 10- 7M) but was not inhibited to any marked extent by isoprothiolane, Vetrazin or a-methyl-DOPA. The effectiveness of diflubenzuron as an inhibitor of chitin synthesis in this system (Iso, 7 x 10- 7 M) correlates well with its potency as a larvicide (LDso, 2�1 x 10- 6 M), providing additional support for the proposal that this compound kills larvae by interfering with chitin deposition in the cuticle. Polyoxin-D was much more effective as an inhibitor of chitin synthesis (Iso, 6 x 10- 7 M) than as a larvicide (LDso, 2�0x 10- 5 M). It was established that the final intermediate of chitin biosynthesis (UDP-N-acetylglucosamine) was formed in the isolated integuments in the presence of diflubenzuron and polyoxin-D. It seems likely therefore that both compounds interfere with the final polymerization step of the chitin biosynthesis pathway.

Nutrition and reproduction in the Australian Sheep Blowfly Lucilia cuprina.

Carbohydrate, protein, and salts are essential for the development of eggs by Lucilia cuprina (Wied.). In the presence of sucrose, each of the following is adequate for egg development: casein, milk, yeast, egg albumen, and gelatin plus L-tryptophan. However, egg development on these foods is slower than on liver. Contrary to previous findings, it is shown that sheep droppings may provide adequate food for the development of eggs in L. cuprina, especially droppings from sheep on improved pastures during spring and autumn, when both pasture and faeces have a relatively high protein content.

Utilization and digestion of Carbohydrates by the Australian Sheep Blowfly Lucilia cuprina.

The nutritional value of 30 carbohydrates, mainly mono- and oligosaccharides and sugar alcohols, was determined by longevity experiments on the Australian sheep blowfly Lucilia cuprina. Carbohydrates which maintain life for considerable periods are hydrolysed by enzymes present in the gut and salivary glands. The crop is an important site of hydrolysis of carbohydrates. The midgut and salivary glands secrete similar carbohydrases. These include two �-glucosidases, an �-galactosidase, and an amylase. A �-fructofuranosidase may be present, but this requires confirmation. Field experiments indicated that the important natural sources of carbohydrates were the honeydew of coccids and psyllids and the nectar of Eucalyptus blossoms.